Ready-to-use storable methanolic solution of a biocompatible and biocidal polyvinylpyridine polymer

ABSTRACT

An alcoholic solution of a partially quaternarized polyvinylpyridine polymer is provided. The alcoholic solution has biocidal and biocompatibility properties. The alcoholic solution is ready-to-graft in order to confer biocidal properties on various surfaces and to prevent biofilm formation on such surfaces.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/427,464, filed on Nov. 29, 2016, entitled “Alcoholic Solution ofa Partially Quaternarized Polyvinylpyridine,” the entire contents ofwhich are incorporated by reference herein.

BACKGROUND OF THE INVENTION

The present invention generally relates to the field of biocides and, inparticular, to an alcoholic solution of a partially quaternarizedpolyvinylpyridine polymer having both biocidal and biocompatibilityproperties. The present invention is designed to be a ready-to-usesolution for reducing biofilm formation on various surfaces bycovalently grafting a biocidally active copolymer using achloromethylcatechol or iodopropyltrimethoxysilane spacer.

Biocidal polymers are well-known in the art and becoming increasinglyimportant in order to contain and control the spread of infectiouspathogens in a variety of health and industrial applications. To thisend, biocidal polymers have been developed for use in solution form aswell as to incorporate biocidal activity onto materials via coatings.

Therefore, it would be highly desirable to have a solution of a biocidalpolymer having both biocidal and biocompatibility properties for aprolonged storage period. It is, therefore, desirable to have aready-to-use biocidal product which prevents fast reticulation involume, thereby prolonging storage period.

BRIEF SUMMARY OF THE INVENTION

To address shortcomings of prior art, the inventors studied andmeticulously determined the reaction period during whichpolyvinylpyridine (PVP), monomer A and monomer B are reacted in analcoholic solution to prepare a statistical copolymer having bothbiocidal and biocompatible properties.

The present invention, therefore, provides a ready-to-use alcoholicsolution of biocidal copolymer designed to be stable for a prolongedstorage period with no spontaneous volume reticulation. According to thepresent invention, the alcoholic solution comprises a biocidally activepartially quaternarized polyvinylpyridine polymer.

In another aspect, the present invention also provides a one-stepprocess to prepare a ready-to-use alcoholic solution of biocidalcopolymer, which is stable and prevents reticulation in volume duringthe storage period. According to the present invention, the processcomprises a step of reacting polyvinylpyridine (PVP), monomer A andmonomer B in an alcoholic solution for a desired reaction period toobtain a biocidally active partially quaternarized polyvinylpyridinepolymer.

The present invention further provides a one-step process to prepare astatistical copolymer by reacting polyvinylpyridine, monomer A andmonomer B in an alcoholic solution. According to the present invention,monomer A is used in 5% fixed proportion to achieve stability of thesolution and prevent reticulation in volume during the storage period.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention, will be better understood when read in conjunction with theappended drawings. For the purpose of illustration, there is shown inthe drawings an embodiment which is presently preferred. It should beunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities shown.

FIG. 1 represents PM-IRRAS spectra to determine the

$\frac{N^{+}}{N}$ratio using the area ratio between the stretching mode of the C—N⁺ band(1640 cm⁻¹) and C—N band (1600 cm⁻¹);

FIG. 2 represents MRSA (Methicillin-resistant Staphylococcus aureus)bacterial killing at 37° C. in rich medium (Brain-Heart Infusion, BHI)after 1 hour with a 10⁷ CFU/mL inoculum (CFU: Colony Forming Units);

FIG. 3 represents MRSA (Methicillin-resistant Staphylococcus aureus) invitro biofilm (Brain-Heart Infusion, BHI) after 3 days on titaniumplates (spin-coated treated surface vs. control) at 37° C. usingscanning-electron microscopy with field-emission gun with a 10⁷ CFU/mLinoculum (CFU: Colony Forming Units);

FIG. 4 represents MRSA (Methicillin-resistant Staphylococcus aureus) invitro biofilm (Brain-Heart Infusion, BHI) after 24 hours on titaniumplates (spin-coated treated surface vs. control) at 37° C. usingscanning-electron microscopy with field-emission gun with a 10⁷ CFU/mLinoculum (CFU: Colony Forming Units);

FIG. 5 represents a young MRSA (Methicillin-resistant Staphylococcusaureus) biofilm (Brain-Heart Infusion, BHI) after 3 hours on titaniumsurfaces (spin-coated treated vs. control) at 37° C. using Atomic ForceMicroscopy (AFM) with a 10⁷ CFU/mL inoculum (CFU: Colony Forming Units);

FIG. 6 represents a young MRSA (Methicillin-resistant Staphylococcusaureus) biofilm (Brain-Heart Infusion, BHI) after 6 hours on titaniumplates (spin coated treated vs. controls) at 37° C. usingscanning-electron microscopy with field-emission gun with a 10⁷ CFU/mLinoculum (CFU: Colony Forming Units);

FIG. 7 represents a mature MRSA (Methicillin-resistant Staphylococcusaureus) biofilm (Brain-Heart Infusion, BHI) after 7 days on titaniumplates (spin coated treated vs controls) at 37° C. usingscanning-electron microscopy with field-emission gun with a 10⁷ CFU/mLinoculum (CFU: Colony Forming Units);

FIG. 8 represents MC3T3 (murine osteoblast precursor cell line) cellviability (MTT-assay) after a 72-hour contact on control and graftedtitanium of varying surface cationic densities;

FIG. 9 represents L929 (murine fibroblast cell line) cell viability(MTT-assay) after a 72-hour contact on control and grafted titanium ofvarying surface cationic densities; and

FIG. 10 represents L929 (murine fibroblast cell line) cell aspect aftera 72 h culture on titanium plates (spin-coated or controls) usingscanning-electron microscopy with field-emission gun.

DETAILED DESCRIPTION OF THE INVENTION

Certain terminology is used in the following description for convenienceonly and is not limiting. The words “right”, “left”, “lower”, and“upper” designate directions in the drawings to which reference is made.The terminology includes the above-listed words, derivatives thereof,and words of similar import. Additionally, the words “a” and “an” mean“at least one.” Further, the skilled artisan will understand that thepresent invention can be practiced without employing the specificdetails provided herein, or that it can be used for purposes other thanthose described herein. The drawings and descriptions are intended to beexemplary of various aspects of the present invention and are notintended to narrow the scope of the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The term “biocide”, as used herein, means a chemical compound, achemical composition, a chemical formulation which can kill or renderharmless a microorganism exemplified by bacterium, yeast, and fungi.

The term “copolymer” as used herein is defined as a polymer that is madeup of more than one type of monomer.

The terms “halo” or “halogen” by themselves or as part of anothersubstituent, have the same meaning as commonly understood by one ofordinary skill in the art, and preferably refer to chlorine, bromine oriodine atom.

The expression “statistical copolymer” as used herein is defined as acopolymer that is made up of more than one monomer, and in which thedifferent monomer units are randomly distributed in the polymeric chain.Unless stated otherwise, the terms “copolymer”, “biocidal polymer”, and“partially quaternarized polyvinyl pyridine” refer to statisticalcopolymer.

The present invention provides a ready-to-use alcoholic solution of apartially quaternarized polyvinylpyridine polymer having both biocidaland biocompatibility properties, which is stable and preventsreticulation in volume during the storage period.

In particular, the present invention provides an alcoholic solution of abiocidally active polymer and methods of preparing the same. Thesolution comprises a biocidally active partially quaternarizedpolyvinylpyridine polymer obtained by reacting polyvinylpyridine (PVP)with combinations of monomer A and monomer B in an alcoholic solution.It was surprisingly discovered that polyvinylpyridine (PVP), whenreacted with combinations of monomer A and monomer B in an alcoholicsolution for a reaction period of two days, resulted in a statisticalcopolymer that showed biocidal and biocompatibility propertiessimultaneously. The solution was found stable and prevented reticulationin volume during the storage period when monomer A was used in 5% fixedproportion, thus prolonging the storage period.

It was further surprisingly discovered that quaternarization of apyridine nucleus with monomer A was kinetically faster than with themonomer B in alcoholic solution. The quaternarization with the monomer Bled to a

$\frac{N^{+}}{N}$ratio, which was found to be dependent on the reaction period. Theinventors studied and found a specific

$\frac{N^{+}}{N}$ratio and reaction period which resulted in a statistical copolymer thatshowed both biocidal and biocompatibility properties simultaneously.

According to the embodiments of the invention, the specific

$\frac{N^{+}}{N}$ratio is 44±2% and the reaction period is two days for obtaining astatistical copolymer having biocidal and biocompatibility propertiessimultaneously.Statistical Copolymer

The statistical copolymer according to the present invention accordingto scheme 1 or scheme 2 contains a monomer A to immobilize the polymericchain on the activated surface through a covalent bond between thesurface and the polymeric moiety and a monomer B to obtain a specific

$\frac{N^{+}}{N}$ratio and improve biocompatibility.

The monomer A is selected for its ability to anchor to the activatedsurface. According to preferred embodiments of the invention, themonomer A that may be part of the statistical copolymer preferablyincludes iodopropyltrimethyoxysilane (used in scheme 1) orchloromethylcatechol (used in scheme 2). The monomer A is preferablyused in 5% fixed proportion to achieve stability of the solution andprevent reticulation in volume during the storage period.

According to preferred embodiments of the invention, the monomer B thatmay be part of the statistical copolymer includes a butyl chain,preferably bromobutane. According to a preferred embodiment of theinvention, the statistical copolymer is prepared by a one-step reactionprocess in an alcoholic solvent. The solvent used in the presentinvention includes methanol and ethanol. In a preferred embodiment ofthe invention, methanol is used as a preferred solvent.

In a preferred embodiment, the one-step reaction process to obtain thestatistical copolymer is carried out in a methanolic solvent for twodays, which results in a specific

$\frac{N^{+}}{N}$ratio of 44±2%.

The biocidal polymer of the present invention can be used on varioussurfaces including titanium and titanium alloys, iron, ceramics, steel,polyethylene (low and high reticulation for use in biomedical implants,after prior plasma activation), teflon (after prior plasma activation),polyethylene terephthalate (after prior plasma activation), andpolypropylene (low and high density, after prior plasma activation),cotton, silicon, wood, glass, and all cellulosic compounds. It will beunderstood that this is a non-exhaustive list. The biocidal polymer ofthe present invention can be used to contain and control the spread ofinfectious pathogens in a variety of health and industrial applications.

The following examples are included solely to provide a clearer and moreconsistent understanding of the present invention.

Example 1 (FIG. 1)

Synthesis of a Statistical Copolymer Using Iodopropyltrimethoxysilane

Polyvinylpyridine (PVP) was reacted with iodopropyltrimethoxysilane (50mEq) and bromobutane (1 Eq) in boiling methanol in a one-step processfor a reaction time of two days to obtain biocidal and biocompatibilitysimultaneously (48 hours,

$ {\frac{N^{+}}{N} = {44 \pm {2\%}}} ).$

For 12 hours,

$\frac{N^{+}}{N} = {12 \pm {2{\%.}}}$

For 36 hours,

$\frac{N^{+}}{N} = {31 \pm {2{\%.}}}$

For 48 hours,

$\frac{N^{+}}{N} = {44 \pm {2{\%.}}}$

For 96 hours,

$\frac{N^{+}}{N} = {45 \pm {3{\%.}}}$

$\frac{N^{+}}{N}$ratio was determined through PM-IRRAS spectra using the area ratiobetween the stretching mode of the C—N⁺ band (1640 cm⁻¹) and C—N band(1600 cm⁻¹).

It can be seen that the rates of quaternarization after 48 hours or 96hours of reaction time in refluxing methanol are the same within theaccuracy of measurement. Using PM-IRRAS spectra following deposition andcopolymer grafting on titanium, it was observed that quaternarizationincreased with reaction time (12 h to 96 h) (FIG. 1).

Example 2

Grafting of Titanium Surface

The solution of statistical copolymer as prepared using scheme ofExample 1 was distributed and covalently grafted on a polished (4000grit paper) and activated titanium (piranha solution or plasmaactivation) surface using an iodopropyltrimethoxysilane chain.

Example 3

Synthesis of a Statistical Copolymer Using Chloromethylcatechol

Polyvinylpyridine (PVP) was reacted with chloromethylcatechol (20 to 50mEq) and bromobutane (1 Eq) in boiling methanol in a one-step processfor a reaction time of two days to obtain biocidal and biocompatibilityproperties simultaneously.

For 48 hours,

$\frac{N^{+}}{N} = {44 \pm {2{\%.}}}$

$\frac{N^{+}}{N}$ratio was determined through PM-IRRAS spectra using the area ratiobetween the stretching mode of the C—N⁺ band (1640 cm⁻¹) and C—N band(1600 cm⁻¹).

Example 4

Grafting of a Titanium Surface

The solution of statistical copolymer as prepared by Example 3 wasdistributed and covalently grafted on the activated titanium surfaceusing a chloromethylcatechol chain.

Example 5 (FIG. 2)

Deposition of the Copolymer on a Titanium Surface

Grafted titanium surfaces showed a high bactericidal effect in vitroagainst methicillinresistant Staphyloccocus aureus (MRSA) within 1 hourunder proliferating conditions (37° C., B.H.I, Brain Heart Infusionmedium). The effect depended on the thickness of the grafted layer. Spincoated (8 nm monolayer) surfaces showed an average 99% bactericidalactivity in vitro within 1 hour. Thicker coatings achieved 100%sterilization of surfaces within 1 hour. Inhibition growth activityagainst MRSA showed a 1.6 log₁₀ inhibition with spin coated surfaces andstill 100% sterilization with thicker coatings. The bars in FIG. 2.display control and treated plates bacterial counts following 1-hourcontact with a 20 μL bacterial suspension (107 CFU/ml inoculum) appliedon the surface with a cover-slip.

Example 6 (FIG. 3, FIG. 4, FIG. 5, FIG. 6, FIG. 7)

Deposition of the Copolymer on Titanium

Grafted titanium surfaces showed a high in vitro anti-biofilm activityagainst MRSA under proliferating conditions (37° C., Brain HeartInfusion, Medium Change every 24 hours, 10⁷ CFU/mL inoculum) up to 7days. Scanning Electron Microscopy with Field Emission Gun (SEM-FEG)findings showed a membrane perforation of bacteria, bacterial shrinkage,reduction of adherent bacteria and biofilm thickness. FIGS. 3-4 showbacterial perforation and shrinkage on the coated titanium. FIG. 5 showsbacterial perforation and volume shrinkage on the treated surfaces. FIG.6 shows low bacterial surface density on the treated titanium. FIG. 7shows low bacterial surface density on the treated titanium andmulti-layer bacterial aspect on the control titanium. Atomic forcemicroscopy findings confirmed these morphologic modifications.

Example 7 (FIG. 8, FIG. 9, FIG. 10)

Determination of Biocompatibility Property

L929 (murine fibroblast cell line) cells were cultured in DMEM(Dulbecco's Mod Eagle Medium) supplemented with 10% FBS (fetal bovineserum), 2 mM glutamin, 100 mug/mL streptomycin and 100 mug/mLpenicillin. MC3T3 (murine osteoblast precursor cell line) cells werecultured in MEM-α (Gibco Invitrogen, France) enriched with 10% FBS(fetal bovine serum), 2 mM glutamin, 100 U/mL penicillin, and 100 μg/mLstreptomycin. After incubation at 37° C. in humid atmosphere and 5% CO₂,a trypsin EDTA (ethylenediaminetetraacetic acid) treatment was appliedin order to retrieve all the adherent cells. 5×10⁵ cells of each linewere seeded on each titanium plate in a volume of 1 mL of theirrespective culture medium within 24-well cell culture plates. Then, anMTT-assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)was performed by comparing the optical densities of the studied media at550 nm in order to deduce cell survival at 72 hours. Interestingly,copolymers synthesized according to the same process in only 12 hourswith no lateral butyl chain did not show biocidal activity, butsignificantly enhanced osteoblast adhesion and survival at 72 hours.Such coatings might be of interest to facilitate implantosteointegration.

Referring to FIG. 8, MC3T3 cell viability decreased with higher surfacecationic densities (past 12 hour reaction time). Cationic densities wererelated with reaction time. Optical density was 550 nm. If the reactiontime was 12 hours, cells deposited on grafted titanium surfacespersistently showed higher cell survival at 72 hour than cells oncontrol titanium plates. If the reaction lasted 4 days, survivaldecreased to the critical level of viability.

Referring to FIG. 9, L929 (murine fibroblast cell line) cell viabilitydecreased with higher reaction time or surface cationic densities.Surface cationic densities were related with reaction time. L929 cellsshowed better tolerance of higher surface cationic densities comparedwith MC3T3 cells. Optical density was 550 nm.

Referring to FIG. 10, a similar morphological aspect and density wasobserved.

SEM-FEG (Scanning-electron microscopy with field emission gun) imageswere obtained using polished control titanium plates or titanium platesgrafted with the described copolymer synthesized in 2 days of reactiontime. Cells had similar morphologic patterns on control and graftedplates.

In conclusion, biocompatibility assessments (MTT-assay to determine cellmetabolic activity) showed a good to excellent cell viability on twocell lines (fibroblasts L929 and preosteoblasts MC3T3) after 72 hours invitro, depending on surface cationic density. A higher cationic densityresulted in a decreased survival at 72 hours.

Example 8

A potential application against wood-boring insects is grafting thecopolymer with a maximum reaction yield on wood cellulose. The mechanismwould be direct or indirect. The direct mechanism comprises grafting abiocidal copolymer on cellulose in order to prevent cellulose digestionby cellulose-eating insects' digestive enzymes (such as Cx-cellulase orothers). The indirect mechanism consists of killing the protozoa,bacteria or fungi that are responsible for the production of suchenzymes (such as C1-cellulase or others) in cellulose-eating insects'gastrointestinal system.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

We claim:
 1. An alcoholic solution comprising a polyvinylpyridinepolymer of formula I or formula II:

wherein X is 12±2% to 45±2%.
 2. The alcoholic solution of claim 1,wherein the polymer of formula I is a copolymer of obtained by reactingpolyvinylpyridine, chloromethylcatechol and bromobutane.
 3. Thealcoholic solution of claim 2, wherein the copolymer is prepared by aone-step process reaction in an alcoholic solvent, wherein the alcoholicsolvent is one of methanol and ethanol, and wherein the one-step processreaction is carried out for two days.
 4. The alcoholic solution of claim1, wherein the polymer of formula II is a copolymer obtained by reactingpolyvinylpyridine, iodopropyltrimethoxysilane and bromobutane.
 5. Thealcoholic solution of claim 4, wherein the copolymer is prepared by aone-step process reaction in an alcoholic solvent, wherein the alcoholicsolvent is one of methanol and ethanol, and wherein the one-step processreaction is carried out for two days.
 6. The alcoholic solution of claim1, wherein X is 44±2%.
 7. The alcoholic solution of claim 6, wherein thepolyvinylpyridine polymer of formula I or formula II is biocompatible.8. The alcoholic solution of claim 3, wherein X is 44±2%.
 9. Thealcoholic solution of claim 8, wherein the polyvinylpyridine polymer offormula I biocompatible.
 10. The alcoholic solution of claim 5, whereinX is 44±2%.
 11. The alcoholic solution of claim 10, wherein thepolyvinylpyridine polymer of formula I biocompatible.